1. Field of the Invention
The present invention relates to a liposomal preparation, its production and use. Areas of application are pharmacy and medicine, particularly its use as carrier systems for drug substances, vaccines, diagnostics and vectors.
2. Brief Description of the Background of the Invention Including Prior Art
Liposomes have gained increasing importance in the past years as carriers or encapsulation means for various substances, particularly in medical applications (see Liposomes from Physics to Applications, D. D. Lasic, Elsevier, Amsterdam 1993). Among the various types of liposomes mostly those are regarded favorable, which have a small to intermediate vesicle size such as from about 10 to 300 nm with good size uniformity and which have one single membrane shell and provide a unilamellar vesicle. Moreover, the active substances should be entrapped or incorporated in the liposomes in an efficient manner.
For the preparation of liposomes, numerous methods have been described. These methods, however, fulfill only insufficiently at least one of the above mentioned main requirementsxe2x80x94small to intermediate liposome size or high encapsulation efficiency. This is mainly the case when the active substance is water soluble and thus should be encapsulated and entrapped in the aqueous interior of the liposomes. The so-called DRV-method and the so-called REV-method both yield good encapsulation efficiencies but at the same time yield populations of inhomogeneous, mostly large liposomes. In contrast, other previously reported preparation methods based on ultrasonic treatment, high-pressure homogenization or detergent removal and dialysis can achieve in case of homogeneous, small to intermediate sized liposomes only a poor encapsulation efficiency, particularly with water soluble substances (Liposome Technology 2nd edition, volume I-III, G. Gregoriadis (Ed.), CRC Press Inc., Boca Raton, Flo., 1993, 37-48; D. Bachmann et al. Preparation of Liposomes using a Mini-Lab 8.30 H high-pressure homogenizer, Int. J. Pharm. 91, 1/93, 69-74).
Liposomes, after three decades of research, are still gaining increasing interest with special emphasis more recently on their use as drug carrier systems. For therapeutic purposes, they must be loaded with active substances. This is more easily achieved with lipophilic or amphiphilic molecules as they have a tendency to be incorporated in the liposomal membrane. In contrast, hydrophilic molecules must be encapsulated in the aqueous interior which, in general, cannot easily be performed in an efficient manner.
A variety of liposome preparation techniques have been developed over the past three decades, none of them, however, perfectly fulfilling the two basic requirements homogeneous and not too large liposome sizes and efficient encapsulation of hydrophilic molecules at the same time. Whereas the dehydration-rehydration vesicles DRV techniques (C. Kirby and G. Gregoriadis, 1984, Dehydration-rehydration vesicles: a simple method for high yield drug entrapment in liposomes, Biotechnology, pp. 979-984), and reverse-space evaporation techniques REV (F. Szoka and D. Papahadjopoulos, 1978, Procedure for preparation of liposomes with large internal aqueous space and high capture by reverse-phase evaporation, Proc. Natl. Acad. Sci., USA 75, 4194-4198), achieve high encapsulation efficiencies but only relatively large and heterogeneous liposome sizes, it is just the opposite with high-pressure homogenization (M. Brandl, D. Bachmann, M. Drechsler, and K. H. Bauer, 1990, Liposome preparation by a new high-pressure homogenizer Gavlin Macron Lab 40 Drug Dev. Ind. Pharm. 16, 2167-2192; M. Brandl, D. Bachmann, M. Drechsler, and K. H. Bauer, 1993, Liposome Preparation using High-pressure Homogenizers, in G. Gregoriadis (Ed.) Liposome Technology 2nd edition, Vol. 1, pp. 49-65, CRC, Boca Raton), or detergent depletion techniques (J. Brunner, P. Skrabal, and H. Hauser, 1976, Single bilayer vesicles prepared without sonication, physico-chemical properties, Biochim. Biophys. Acta 455, 322-331). The concept at the beginning of the invention was fairly straightforward: for low-molecular weight hydrophilic molecules, which represent the majority of classical drug substances, the degree of entrapment into liposomes can be regarded a result of partition of the aqueous drug solution at the moment of liposome formation into a compartment inside the liposomes and a compartment in-between the liposomes.
The ratio of volume inside the liposomes compared to the total aqueous volume of the preparation is defined as encapsulation efficiency. The ratio can theoretically be increased by two alterations within the system: Firstly, when at constant lipid concentration, only a few large liposomes are formed instead of many small liposomes, the entrapped aqueous volume is increased. For technical, and in the case of i.v. injection, also for pharmacokinetic reasons, the enlargement of liposome size is confined to narrow limits. In the case of i.v. injection liposomes sizes between 70 and not more than 200 (D. Liu, A. Mori, and L. Huang, 1992, Role of liposome size and res. blockade in controlling biodistribution and tumor uptake of gml-containing liposomes, Biochem. Biophys. Acta 1104, 95-101), and sufficient size homogeneity (D. Liu and L. Huang, 1992, Size homogeneity of a liposome preparation is crucial for liposome biodistribution in vivo, J. Liposome Res. 2, 57-66), are regarded as favorable for most applications. Secondly, with increasing lipid concentration, more liposomes per unit volume of the preparation are formed. This again should lead to an increase in the ratio of aqueous space inside compared to in-between the liposomes as long as liposome shape and lamellarity are unchanged. Phospholipid, however, when dispersed in aqueous medium at or above lipid contents of 200 to 300 mM result in highly viscous dispersions up to semisolid consistency. Although it was expected that these viscous to semisolid preparations would no longer be liposome dispersions, it was of interest to prepare such xe2x80x9cpastesxe2x80x9d preferentially with homogeneous physicochemical characteristics. Based on previous experience with liposome preparation by the one-step technique (M. Brandl, D. Bachmann, M. Drechsler, and K. R. Bauer, 1990, Liposome preparation by a new high-pressure homogenizer Gavlin Macron Lab 40 Drug Dev. Ind. Pharm. 16, 2167-2192, M. Brandl, D. Bachmann, M. Drechsler, and K. H. Bauer, 1993, Liposome Preparation using High-pressure Homogenizers, in G. Gregoriadis (Ed.) Liposome Technology 2nd edition, Vol. 1, pp. 49-65, CRC, Boca Raton), high-pressure homogenization for xe2x80x9cforced hydrationxe2x80x9d of lipids was employed.
In order to study the inner structure of the pastes in terms of homogeneity, freeze fracture electron microscopical visualization was used. It was noticed that these pastes may retain hydrophilic markers and thus potentially serve as depot formulations for controlled release of drugs. The drug release behavior of the pastes was further analyzed in a standard in-vitro continuous flow through apparatus as normally employed for release tests of ointments. Preliminary reports of this project have been published recently, M. Brandl and R. Reszka, 1995, Preparation and characterization of phospholipid membrane gels as depot formulations for potential use as implants, Proc. Intern. Symp. Control. Rel. Bioac. Mater. 22, 472-473; M. Brandl, C. Tardi, M. Menzel, and R. Reszka, 1995, Highly concentrated phospholipid dispersions: preparation by high pressure homogenization and analysis of drug release. Proceedings 4th Liposome Res. Days. Freiburg PSI (not completely legible in publication); M. Brandl, D. Bachmann, R. Reszka, and M. Drechsler, 1996, Unilamellar Liposomal Preparations with High Active Substance Content.
1. Purposes of the Invention
The goal of the invention is to make a liposomal preparation with good encapsulation efficiency for active substances, based on homogeneous liposomes of small to intermediate size.
These and other objects and advantages of the present invention will become evident from the description which follows.
2. Brief Description of the Invention
The present invention provides a liposomal preparation comprising homogeneous unilamellar liposomes of small to intermediate size, having a vesicular structure in tight packing, and forming carriers of active substances, and at least 20% by weight relative to the total weight of the liposomal preparation of an active ingredient in entrapped form inside the homogeneous unilamellar liposomes, wherein the liposome preparation forms a highly viscous liposome gel.
The liposomal preparation preferably contains more than 95 percent by weight of the homogeneous unilamellar liposomes exhibiting a diameter of from about 10 to 300 nm, and has the active substances as members selected from the group consisting of drug substances, vaccines, diagnostics, vectors, and mixtures thereof.
According to the invention a liposomal preparation is produced comprising the steps of subjecting a mixture comprising membrane-forming amphiphiles, solvent and an active ingredient, which active ingredient is to be encapsulated, to a high-pressure homogenization under pressures of from about 50 to 1600 bar (5-160 MPa), subsequently removing the solvent, subsequently freezing the mixture, thawing the mixture, and transferring the mixture into a freely flowing dispersion. Furthermore a step of extruding the mixture through filters having a pore width of from about 0.1 to 1 xcexcm can follow.
Preferably the solvent employed is removed by evaporation or by spray drying. The subjecting to the high-pressure homogenization can be preceded by a preparation of a thin, dry lipid film of the membrane-forming amphiphiles. The solvent is preferably water. Advantageously, the mixture is subjected at least two times and not more than 50 times to the high-pressure homogenization.
The membrane-forming amphiphiles can be members selected from the group consisting of lipids, phospholipids of natural orogin, phospholipids of synthetic origin, synthetic amphiphiles, and mixtures thereof. The lipid can be cholesterol. The phospholipid can be a member selected from the group consisting of phosphatidyl choline, phosphatidyl glycerol and mixtures thereof. The synthetic amphiphiles can be reactants of members selected from the group consisting of block-copolymers, alkyl esters, alkyl ethers and alkyl amides and mixtures thereof formed with members selected from the group consisting of alcohols, diols, triols, polyols, amines, amino acids, peptides, saccharides and mixtures thereof.
A liposomal preparation can be applied, which comprises homogeneous unilamellar liposomes of small to intermediate size, having a vesicular structure in tight packing, and forming carriers of active substances; and at least 20% by weight relative to the total weight of the liposomal preparation of an active ingredient in entrapped form inside the homogeneous unilamellar liposomes, wherein the liposome preparation forms a highly viscous liposome gel, for effecting a release of liposomes in aqueous medium under retention of a high proportion of the active ingredient present in entrapped form. At least 20% of the active ingredient can be present in entrapped form.
The invention furnishes further a liposomal preparation, consisting of liposomes as carriers of active substances such as drug substances, vaccines, diagnostics, as well as vectors, characterized in that it consists of homogeneous unilamellar liposomes of small to intermediate size, preferably having a diameter of 10 to 300 nm, having a vesicular structure in tight packing, and it contains at least 20% of the active ingredient in entrapped form and represents a highly viscous liposome gel.
A mixture of membrane-forming amphiphiles, water and active ingredient is to be encapsulated and is subjected one time or several times, at the most fifty times, to a high-pressure homogenization with pressures of from 50 to 1600 bar (5-160 MPa), if necessary after preparation of a thin, dry lipid film and subsequent removal of the solvent by evaporation or spray drying, and wherein there occurs subsequently, if necessary, a treatment by freezing and thawing, and, if suitable, a transfer into a freely flowing dispersion and subsequently, if suitable, an extrusion through filters having a pore size of 0.1 to 1 xcexcm. Lipids, phospholipids of natural or synthetic origin or synthetic amphiphiles are employed as membrane-forming amphiphiles. Preferably cholesterol is employed as lipid. Preferably, phosphatidyl choline or phosphatidyl glycerol is employed as phospholipid. Block-copolymers, alkyl esters, alkyl ethers and alkyl amides of alcohols, diols, triols and polyols, amines, amino acids, peptides, and saccharides can be employed as synthetic amphiphiles. The liposomal preparation can be applied for release of liposomes in aqueous medium under retention of a high proportion, preferably at least 20%, of the active ingredient in entrapped form.
The preparation of highly concentrated dispersions of phosphatidyl choline PC via high-pressure homogenization was described above. The obtained semi-solid matrices retained incorporated hydrophilic markers for time periods of at least several hours and thus appeared suitable as depot formulations for drugs. During in-vitro release tests, markers were found to be released in free as well as in liposome-entrapped form (compare M. Brandl, C. Tardi, M. Menzel, R. Reszka, Proc. 4th Lipos. Res. D., 1995).
Applicants investigated whether the release kinetics of the incorporated marker can be influenced by the concentration of phosphatidyl choline PC in the matrix. Applicants found that the overall tendency was a decrease of release rate with increasing lipid concentration for lipid concentrations between 30% and 60% (m/m). When comparing lipid concentrations of 40% and 50% (m/m), however, a dramatic change was found. Whereas 40% (m/m) matrices showed mean release rates of about 15% of total incorporated marker per hour and durations of about 6 hours until 100% release was achieved, the 50% (m/m) matrices released only 2% per hour in mean and release continued for more than 48 hours. The fraction of the marker released in liposome-entrapped form was also much smaller with the more concentrated matrix. None of the matrix was left in the release cell after the end of marker release from the 40% (m/m) preparation. In contrast with 50% (m/m) matrices, the cell still contained a remainder of the lipid matrix, which had been depleted of all marker.
These results indicate that structural changes of the matrices occur at a critical lipid concentration of from about 30% to 60%, and preferably from 40% and 50% (m/m). This observation agrees with results of freeze-fracture electron microscopical examination of the fine structure of the matrices. Matrices consisting of 40% or less of lipid showed structures of densely packed small unilamellar vesicles (SUVs) throughout. In contrast, matrices of lipid concentrations of 50% and above showed various structural elements besides SUVs, large multilamellar vesicles and planar staples of bilayers were also found. It is conceivable now that the described three-dimensional liposomal networks below the critical concentration solely consist of SUVs and thus fully erode by continuous xe2x80x9cbudding offxe2x80x9d of intact liposomes. Above this critical concentration on the other hand, only a proportion of SUVs is embedded in less defined structures which do not erode easily. The marker in this case is released substantially exclusively via diffusion. In conclusion, raising the lipid concentration does not only affect the release rate but beyond a certain limit also causes a change in release mechanism.
Furthermore, highly concentrated, semi-solid phospholipid dispersions have been investigated. Their preparation is based onxe2x80x9cforced hydrationxe2x80x9d of (phospho-)lipid(s) by high pressure homogenization in the presence of relatively low amounts of water. The inner structure of the obtained semisolid pastes, as revealed by freeze-fracture electron microscopy, can be best described as a matrix of densely packed vesicles. Depending on the lipid content, the characteristics of these vesicles range from very homogeneous, small and unilamellar to more heterogeneous in size as well as lamellarity. Although not comparable to xe2x80x9cclassicalxe2x80x9d liposome dispersions, these multivesicular pastes may be useful as drug carriers. Results from in-vitro release tests demonstrate that they may serve as local depots for controlled release of active compounds. Two release mechanisms are observed occurring at the same time: (1) release of free active molecules via diffusion out of the matrix and (2) budding off of active compound-carrying liposomes from the matrix. Release type and rate are determined among other factors by the phospholipid content of the matrices and thus by their inner structure.